The regulation of intestinal lipoprotein assembly and secretion is poorly understood. Results from our previous studies and those of others suggest that lipid availability regulates lipoprotein synthesis and secretion. Unesterified and esterified cholesterol are important components of the surface and core of the triacylglycerol-rich lipoprotein particle, respectively. The contribution of newly-synthesized cholesterol, micellar cholesterol, or plasma membrane cholesterol to the normal assembly of the lipoprotein, however, is unknown. The investigators are postulating that it is cholesterol derived from the plasma membrane which is primarily utilized for lipoprotein assembly within an intestinal cell. It is also postulated that plasma membrane cholesterol content regulates cellular cholesterol metabolism and that normal cholesterol trafficking is required for this regulation to occur. P-glycoprotein, a protein located on the plasma membrane whose function in normal intestine is unknown, is postulated to play a role in cholesterol trafficking and cholesterol metabolism. This proposal will delineate experiments which will address cholesterol trafficking in intestinal cells and define the regulation of this process. The regulation of cholesterol metabolism at the level of gene expression by changes in plasma membrane cholesterol and cholesterol trafficking will be investigated. The contribution of newly-synthesized cholesterol, micellar cholesterol, and plasma membrane cholesterol to the unesterified and esterified cholesterol content of triacylglycerol-rich lipoproteins will be studied. Regulation of lipoprotein assembly and secretion by availability of newly-synthesized cholesterol and cholesterol esters will be addressed. Lastly, the role of p-glycoprotein in cholesterol trafficking, cholesterol metabolism, and lipoprotein secretion will be studied. These experiments will be performed in CaCo-2 cells cultured on permeable supports separating an upper and lower chamber. Lipoprotein synthesis and secretion will be estimated by lipid synthesis and secretion, apoB mass secretion, synthesis and degradation of labeled apoB, and apoB mRNA abundance. The regulation of HMG-CoA reductase will be addressed by estimating reductase protein by Western blotting, mRNA abundance, and synthesis and degradation of the protein. ACAT regulation will be estimated by changes in gene expression. To address the role of p-glycoprotein in cholesterol trafficking and metabolism, the investigators will over-express p-glycoprotein in CaCo-2 cells by stable-transfection with human mdr (p-glycoprotein) cDNA and experiments are proposed in gut sacs prepared from mdr1-deficient mice.